PROJECT SUMMARY Small numbers of neurons can play outsized roles in brain function. For example, neuromodulatory circuits that contain only thousands of neurons project throughout the brain to control the activity of billions of neurons in other brain regions. These neuromodulatory circuits, which secrete the neuromodulators dopamine, serotonin, norepineprhine, acetylcholine, and histamine, regulate essential brain functions such as learning, eating, mating, and socialization. Importantly, alteration of neuromodulatory circuit function underlies addiction, e.g. dopaminergic, serotonergic, and noradrenergic functions are potentiated by cocaine and amphetamines, and cholinergic functions by nicotine. However, very little work has addressed how neuromodulatory circuits respond in real time to environmental stimuli, behavioral output, or drug use. In addition, little is known about how these neuromodulatory circuits develop as animals mature. In the cortex and hippocampus, genetically encoded calcium indicators (GECIs) and two-photon microscopy together have been enormously successful in relating activities of specific types of neurons to various stimuli or behavioral outputs. In contrast, efforts to apply calcium imaging to neuromodulatory circuits have proceeded on a far smaller scale, mostly due to their difficult anatomy. The deep locations of neuromodulatory nuclei near other vital brain regions make the placement of optical lenses or fibers very difficult to perform without creating severe behavioral or functional deficits. Thus, what is needed, and what does not exist, is a way to non- invasively record the total the activity of a specific neuromodulatory network from outside the animal. With the long-term goal of enabling noninvasive activity recording of specific neuronal circuits in living animals, we propose to create and implement GECIs that operate not via fluorescence but via bioluminescence, in which a luciferase enzyme reacts with a chemical substrate to produce light in a calcium-dependent manner. The lack of endogenous luciferases leads to background that is negligible, so high contrast can be achieved even with external detectors. In preliminary work, we have generated a calcium-dependent variant that demonstrates 7-fold increase in photonic output in response to calcium, a similar response ratio to the widely used fluorescent reporter GCaMP3. Compared to other bioluminescent calcium reporters, this reporter produces orders of magnitude more photons above 600 nm, to which tissue is relatively transparent. This reporter, which we name red calcium- modulated bioluminescent indicator (orange CaMBI), has been used successfully in cultured neurons. We now propose to develop methods to non-invasively visualize the activity of specific neuromodulatory circuits in freely behaving mice, by (1) testing the ability of CaMBI expressed throughout the brain and substrates injected intravenously, intraperitoneally, intraventricularly to produce luminescence from the brain in an activity- dependent manner, (2) making a transcranial head-mounted optical recording device, and test the ability to detect activity in neuromodulatory circuits, and (3) futher improving CaMBI for brightness and calcium responsiveness.